Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive. • All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.• Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

to the preceding technologies such as zinc-finger nucleases and TALENs [2] .In Streptococcus pyogenes , the adaptive immune system molecule SpCas9 binds gRNA via trans-activating CRISPR (tracr) RNA and scans DNA until it matches gRNA leading to the doublestranded cleavage of the target DNA three nucleotides upstream of the NGG sequence [3] .This latter sequence is called protospacer adjacent motif (PAM) and is the key determinant of the binding specificity [4] .Thus, using spacer arrays containing different gRNAs acquired from previous viral infections, bacteria are capable to recognize and neutralize infecting bacteriophages resembling those previous viral strains [5] .Scientists further developed and improved this system, shortening and combining the tracrRNA containing in repeats of these arrays with artificial gRNAs [6] .Such single guide RNA (sgRNA) can be synthesized from strong polymerase III promoters ( H1 or U6 ) and efficiently guide Cas9 to their targets [7] .
Nowadays, gRNAs can be predicted by different web-based tools [8] .However, not every putative gRNA exhibits the predicted on-target activity, since (i).different cell types may exhibit varying gRNA efficacy [9 , 10] due to the epigenetic status of the target locus [11] .For example, methylation status can impair Cas9 activity and subsequent DNA repair by altering local chromatin structure [12] .(ii).Furthermore, the cell cycle phase of the target cell could also influence the effectiveness of gRNA [13 , 14] .(iii).Genomic stability differences in various cell types might also contribute to gRNA effectivity [9 , 15-18] .(iv).Another important consideration about gRNA efficiency is the DNA sequences flanking the PAM.Such context differences might impact both binding and cleavage efficiency of Cas9 due to its "sliding " towards PAMs, thus profoundly impacting gRNA activities [19] .(v). gRNAs designed using computer models may sometimes encounter challenges related to secondary structure and stability, which could potentially result in suboptimal design quality.As such, unstable sgRNAs may fail to effectively bind to Cas proteins [20] .(vi).In this regard, for example, enrichment of guanines and depletion of adenines renders sgRNAs to be more stable [21] .
Therefore, before experimental use, especially in vivo , it is crucial to assess their cleavage efficiency at the target locus and identify the most efficient ones in vitro .Ideally, the efficiency of such targeting should be analysed within the intact genomic context with spared upstream and downstream sequences, in cells of the same epigenetic context (specific both for the stage of ontogenesis as well as tissue/cell specificity).Only these two contextual conditions might guarantee the effective translation to the in vivo state.However, it is not always possible -as in case of in situ CRISPR-Cas9 techniques [22 , 23] -or practical to fulfill such strict criteria.Scientists employ various assays to validate predicted gRNAs, including the single-strand annealing (SSA) assay, T7 endonuclease I (T7EI) assay, Sanger sequencing, and western blot [24][25][26][27] .The SSA assay combines luciferase analysis to enable high-throughput evaluation of nuclease activity, while the T7EI assay utilizes the structure-selective enzyme T7 endonuclease I to detect DNA structural deformities, providing a simple and cost-effective method for assessing genome targeting efficiency [28] .Sanger sequencing with inference of CRISPR edits (ICE, [29] ) directly examines gRNA target efficiency with precise results, but it is not scalable for screening of on-target efficiencies in multiple gRNAs [26] and cannot be used for in situ CRISPR-Cas9 technique [22 , 23] .Indeed, the latter alters genomes of multiple cells in a defined population producing different indels and other types of edits, but does not produce a single clone for further in vitro or in vivo application [30] .Moreover, Sanger sequencing is relatively costly and operationally complex [31] .Similarly, western blot, while being a simple and widely used technique, is less precise for determining gRNA targeting efficiency and involves the extraction of whole-cell proteins and analysis of target protein expression levels [27] .All these methods are time-consuming, involve multiple steps and sometimes cannot produce solid results.For example, the SSA technique is very effective, but has been used mostly for TALENs and ZFNs, involves co-transfection with target plasmids and nucleases and requires longer time to perform [24] .GUIDE-seq, similar to Sanger sequencing and SSA assay, takes in consideration the chromosomal context and can assess both on-target and off-target cleavage by introducing specialized DNA adapters or oligonucleotides at the sites of DNA double-strand breaks (DSBs) followed by high-throughput sequencing and analysis of those DNA fragments [32] .However, it also cannot be used for direct on-target efficiency analyses for in situ CRISPR-Cas9 approach for the same reason described above [22 , 23] .Another limitation of GUIDE-seq is that it requires transfection with double-stranded oligodeoxynucleotide (dsODN) tags, not tolerable by some cell types [33] .For example, human hematopoietic stem cells or induced pluripotent stem cells reveal pronounced DNA damage response and undergo apoptosis upon high levels of free DNA ends [34] .
A successful CRISPR gene editing application depends greatly on the selection of highly efficient gRNAs.Our aim was to develop an efficient and rapid all-in-one system to check gRNA on-target efficiency in less time with more precision.Here, we describe Noodles, a versatile plasmid, which can benefit researchers and biotechnologists working with CRISPR-Cas9 system.

Principle of the noodles system
To rapidly and sensitively analyze the on-target efficiency of CRISPR gRNAs, we developed the all-in-one plasmid that we designated here as Noodles.In analogy with the predecessor plasmid [1] the system uses advantage of the homology recombinationdependent repair of the split luciferase gene and the resulting luminescence as a readout for CRISPR gRNA-dependent nuclease cleavage of the target region by spCas9 ( Fig. 1 , [Please insert a reference to our Suppl.data file here]).Since the firefly luciferase is split by the cassette containing < in-frame repeat > < stop codon > < gRNA response sequence > < in-frame repeat > , cleavage of the response sequence by Cas9 restores the luciferase gene via single-strand annealing repair mechanism.In contrast to the analogous plasmids [1] , our system comprises the following essential components: Noodles, the all-in-one system for analysis of CRISPR gRNA on-target efficiency.
• Firefly luciferase gene expression cassette split by the response sequence-containing cassette, • Renilla luciferase gene expression cassette as a transfection efficiency control in the dual-luciferase assay.

Brief overview of the method
The candidate gRNA pre-selected using the protocol described in section Design of sgRNAs (see below) should be subcloned to the Noodles plasmid via sticky end ligation of the BsmBI-opened Noodles with the pre-annealed phosphorylated ⟨g-oligos ⟩ (sections Phosphorylation and annealing of the oligonucleotides and Subcloning of gRNAs and response sequences to Noodles , Table 1 ) to generate the Control plasmid.The latter also serves as a template for subcloning the response sequence for the candidate gRNA to generate the Tester plasmid.Notably, in contrast to the gRNA subcloning step, this response sequence should contain PAM after insertion.In analogy with the previous step, the AscI/SbfI-opened Control plasmid should be ligated with the pre-annealed phosphorylated ⟨rs-oligos ⟩ (sections Phosphorylation and annealing of the oligonucleotides and Subcloning of gRNAs and response sequences to Noodles , Table 1 ).The original 14,197 bp Noodles is thus converted to 12,053 bp Control and Tester plasmids.The subsequent dual luciferase assay will be described in detail as well (see section Cell transfection and dual luciferase assay ).In addition to the previous use-case examples [22 , 23] , this study also describes an independent validation of the method (see below).
(2) Select the optimal gRNAs based on the best scores provided by the tool, taking into consideration minimization of off-target activities and maximization of the predicted on-target activity, selectivity and specificity.

Table 1
Template sequences of oligonucleotides for subcloning of gRNA and response sequence.Overhangs for sticky ends ligation are indicated with superscript letters.For simplicity, we designate here the oligonucleotides containing gRNA or response sequence in direct orientation as sense, while in opposite orientation as antisense.
‡ , IMPORTANT!Add an additional G (indicated by bold italic letter) after the overhang at the start of the sense gRNA oligonucleotide and additional C at the end of the antisense oligonucleotide, but ONLY if gRNA does not already contain the starting G.This ensures that gRNA can be effectively transcribed by RNA Polymerase III.
§ , PAM (only present in the response sequence oligonucleotides) is outlined by bold letters.IMPORTANT!The N in the PAM in ⟨rs-oligos ⟩ must correspond to the same nucleotide in the PAM of the target gene for the researcher's project.Order the oligonucleotides for subcloning of candidate predicted gRNAs and the corresponding response sequences (use the template format outlined in Table 1 ) and proceed with the next steps (see below).

Procedure
(1) Reconstitute each sense and antisense oligonucleotide in required amount of ddH 2 O to get a 0.1 mM stock solution.
(2) For each ⟨g-oligos ⟩ or ⟨rs-oligos ⟩ (see Table 1 ), mix the sense and antisense oligonucleotides to set up the phosphorylation reaction mix ( Table 2 ).(3) Phosphorylate the oligonucleotide in a thermocycler at 37 °C for 30 min.(4) Perform a denaturation step at 95 °C for 5 min.
(5) Anneal the oligonucleotides by gradually cooling the mixture to 25 °C at a rate of 5 °C per minute.(6) Dilute annealed oligonucleotides 250-fold by adding 2 μl of the reaction mixture to 498 μl of ddH 2 O.

Materials and reagents
• Noodles plasmid.
• Annealed oligonucleotides from the previous step.

Procedure Generation of the control plasmid.
(1) To subclone gRNA to Noodles and thus generate the Control plasmid, prepare the following mixture ( Table 3 ): (2) Incubate the mixture in a thermocycler at 37 °C for 1 hour.

Selection and validation of the control plasmid.
(1) To verify the successful subcloning of the gRNA, perform colony PCR using the respective sense oligonucleotide for ⟨g-oligos ⟩ from section Phosphorylation and annealing of the oligonucleotides as a forward primer and ATCATGGGAAATAGGCCCTC (Table S2) as the common reverse primer with the following cycler conditions: (2) Prepare the following reaction mixture ( Table 4 ): (3) Notably, this Colony PCR strategy allows testing multiple clones in one PCR tube following identification of the positive reaction and repeat of the colony PCR with single clones per tube.However, the simultaneous restriction-digestion is highly efficient resulting in majority of clones being positive.(4) After submerging the toothpick or pipette tip with a colony into the PCR tube with the mix, keep it at 4 °C in a small volume of LB-ampicillin until the PCR results indicate the positive clone that can be grown overnight, validated by Sanger sequencing and used for the next steps.(5) Control plasmid will be used for the Dual luciferase assay, as well as for subsequent steps to generate the Tester plasmid.

Generation of the tester plasmid.
(1) To subclone the response sequence (the same sequence targeted by selected gRNA in your gene of interest, including PAM) to Control plasmid and thus generate the Tester plasmid, prepare the following mixture ( Table 5 ): (2) Incubate the mixture in a thermocycler at 37 °C for 1 hour.
(1) To verify the successful subcloning of the gRNA, perform colony PCR using the respective sense oligonucleotide for ⟨rs-oligos ⟩ from section 2.4 .as a forward primer and ACCACGCTGAGGATAGCGGTG (Table S2) as the common reverse primer with the following cycler conditions: (2) Prepare the following reaction mixture ( Table 6 ): (3) Verify the positive Tester plasmid colony(ies) by Sanger sequencing and proceed to the next step.

Materials and reagents
• HEK-293T cells (see Table S1 for detailed information about reagents).
• Prepare Stop & Glo Reagent before each use.
• Dilute cell lysate with the Passive Lysis Buffer if luminescence intensity is exceeding the luminometer measurement range.
(5) Next day, selected colonies were transferred to ampicillin-containing LB medium for overnight culture at 37 °C followed by miniprep (Qiagen, 27104), analytical restriction digest with BglII, EcoRV, KpnI, and BamHI.(6) The positive clones of the Intermediate plasmid were then validated by Sanger sequencing.will not be similarly blocked.Noodles does not account for epigenetic factors that may impact Cas9 cutting efficiency.Hence, unless the researchers intend to use in situ CRISPR-Cas9 or other techniques not allowing to produce and expand a single clone, the methods like Sanger sequencing, SSA assay or GUIDE-seq represent great alternatives for assessing the gRNA efficiency.

Fig. 1 .
Fig.1.Noodles, the all-in-one system for analysis of CRISPR gRNA on-target efficiency.

Fig. 4 .
Fig. 4. Validation of gRNAs targeting the mouse glucocorticoid receptor gene.(A) Dual-luciferase assay-assisted analysis of on-target activity of gRNAs targeting GR using Noodles.n = 5 (B, C) Independent in vitro verification of the on-target activity of the selected gRNAs by Qpcr, n = 3 (B) and western blot (C).All data are expressed as mean ± SEM. * * * , p < 0.001; * * * * , p < 0.0001 as assessed by unpaired two-tailed Student's t -test.

Table 3
Mixture for simultaneous restriction of the Noodles plasmid and ligation with ⟨g-oligos ⟩ to generate the Control plasmid.

Table 4
PCR mixture for validation of Control plasmid generation.

Table 5
Mixture for simultaneous restriction of the Control plasmid and ligation with the ⟨rs-oligos ⟩ to generate the Tester plasmid.

Table 6
PCR mixture for validation of Tester plasmid generation.